Irrespective of the organism, genome stability requires that the entire DNA be replicated once and only once per cell cycle. The intricacies of regulatory mechanisms that guarantee initiation of replication and prevent premature reinitiation are still being unraveled even in a well-studied system like E. coli. Replication control is better understood in bacterial plasmids. However, plasmid replicons, although prevalent in bacteria, are seldom found in bacterial chromosomes. Exceptions have been found in secondary chromosomes (one with fewer house-keeping genes) of multichromosome bacteria, such as Vibrio cholerae. In this bacterium, the replication origin of chromosome II bears all the hallmarks of well-studied iteron-carrying plasmids, indicating its plasmid provenance, but it exhibits novel features in the locus that controls replication. We are interested in those features to understand how chromosome II replicates in a cell-cycle specific fashion like other chromosomes, rather than throughout the cell cycle, as do plasmids. We show that while plasmids have only iterons in their control locus, chromosome II has a second kind of sites, which are 39-mers and unrelated in sequence to iterons. The 39-mers bind the same initiator protein as do the iterons but they are the primary negative regulators of replication. They enhance handcuffing, a mechanism known to inactivate plasmid origins with iterons. The activity of the 39-mers is, however, dampened by iterons of the control locus. The control iterons thus facilitate replication, a role not known to be operative in plasmid replication. It appears that, in spite of the similarity in the origin region, the chromosome II control system has diverged considerably from the plasmid system. The novel functions of chromosome II might align its replication to the cell cycle. The two chromosomal origins of V. cholerae are distinct, which raises the question how they fire once per cell cycle. We have determined that in spite of the distinct features of the two origins, once-per-cell-cycle replication requires that both the origins be fully methylated at the adenine residues of their GATC sites. For chromosome I, whose origin is similar to the E. coli origin, methylation was found to be dispensable for replication initiation but required to control replication initiation frequency, as is the case in E. coli. For chromosome II, methylation was additionally required for initiator binding to the origin iterons, an essential function in the initiation process per se. Although methylation is widely used to control many DNA transactions, its role in mediating initiator-origin interactions is an unprecedented finding. The requirement of initiator binding also makes methylation an essential function in V. cholerae. Multichromosomal bacteria (unlike such well-studied mono-chromosome bacteria as E. coli and B. subtilis) may offer opportunities to investigate mechanisms for coordinating replication and segregation of the different chromosomes. An initial indication of inter-chromosomal coordination in V. cholerae has been obtained. We have been able to find conditions where replication of one of the chromosomes could be selectively prevented. It appears that preventing chromosome I replication can prevent/delay chromosome II replication but the reverse is not true. Chromosome II is smaller than chromosome I and the delay might allow the two chromosomes to complete replication at the same time, which might facilitate the coordination of their segregation with the cell division. The mechanism of delay remains to be investigated. Compared to our understanding of how chromosomes segregate in eukaryotes, much less is known about how chromosomes segregate in bacteria. Until recently, segregation studies were done primarily in plasmids, where genes dedicated to plasmid partition (par genes) could be found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both the Vibrio chromosomes have their own par genes. We have succeeded in deleting the par genes of chromosome I without causing much segregation defect. Rather, deletion of one the two par genes (parB) promoted replication. A similar finding has also been made in B. subtilis. The two bacteria, B. subtilis and V. cholerae, have diverged more than a billion years ago but both have retained the par genes and use them for similar purposes. The widespread occurrence of par genes near the replication origins suggests that the genes might have a role in connecting replication and segregation. How might the par genes promote replication is under current investigation. Using both the bacterial and yeast two-hybrid systems we are trying to identify proteins that might interact with Par proteins, which might provide a clue as to their mechanism of action. Cell division must await completion of chromosome replication and movement of the two sister chromosomes to opposite cell halves. In eukaryotes, DNA replication is coupled to cell growth and division via tightly regulated cell cycle-dependent mechanisms. The temporal control of cell division is largely unknown in bacteria. Recently, some of the genes involved in sensing glucose concentration in the growth media have been found to regulate cell size in E. coli and in B. subtilis. Mutations in these genes make cells smaller by about 30% but do not change their growth rates. In a collaborative study, we are determining the timing of replication initiation (initiation age) in these smaller cell variants. The initiation age seems to depend on the bacterium in question. In B. subtilis, the age remains unchanged in the mutants, meaning that at the time of initiation, the cell size is smaller in the case of mutants compared to the wild type. In E. coli, however, initiation is delayed until the mutants reach the size at which initiation occurs in the wild type. The delay in initiation is compensated by increase in replication elongation rate, allowing the replication cycle to complete on time. The initiation delay could be avoided by overproducing the initiator protein, DnaA. The rate-limiting component thus appears to be DnaA in E. coli but not in B. subtilis. The timing replication initiation in B. subtilis might be coupled to division through a cell cycle dependent mechanism, as in eukaryotes.